BOAR SPERMATOZOA DEVELOP ABILITY TO BIND TO OVIDUCT EPITHELIUM DURING PASSAGE THROUGH THE EPIDIDYMIS

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1 BOAR SPERMATOZOA DEVELOP ABILITY TO BIND TO OVIDUCT EPITHELIUM DURING PASSAGE THROUGH THE EPIDIDYMIS Thesis submitted by Santiago Torda Peña, Jr. DVM (LSU) in February 2007 for the degree of Master of Tropical Veterinary Science in the School of Veterinary and Biomedical Sciences James Cook University

2 ii STATEMENT OF ACCESS I, the undersigned, author of this work, understand that James Cook University will make this thesis available for use within the University Library and, via the Australian Digital Theses Network, for use elsewhere. I understand that, as an unpublished work, a thesis has significant protection under the Copyright Act and; I do not wish to place any further restriction on access to this work. Santiago T. Peña, Jr. Date STATEMENT OF SOURCES DECLARATION I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution for tertiary education. Information derived from published or unpublished work of others has been acknowledged in the text and a list of references is given. Santiago T. Peña, Jr. Date

3 iii DECLARATION OF ETHICS The research presented and reported in this thesis was conducted within the guidelines for research ethics outlined in the Joint NHMRC/AVCC Statement and Guidelines on Research Practice (1997), the James Cook University Policy on Experimentation Ethics, Standard Practices and Guidelines (2001), and the James Cook University Statement and Guidelines on Research Practice (2001). The research methodology received clearance from the James Cook University Experimentation Ethics Review Committee (Animal Ethics approval number A1007). Santiago T. Peña, Jr. Date

4 iv STATEMENT ON THE CONTRIBUTION OF OTHERS Professor Phillip Summers supervised the research reported in this thesis, carried out the surgical castrations of the boars, provided advice and assistance with the preparation of the thesis and was a co-author on all papers resulting from this thesis. A stipend was provided by the Australian Agency for International Development (AusAID) for the duration of the research candidature. Project costs were met from IRA and Reproduction Service accounts held by Professor Summers. Santiago T. Peña, Jr. Date

5 v ACKNOWLEDGEMENT The LORD bless thee, and keep thee: The LORD make his face shine upon thee, and be gracious unto thee: The LORD lift up his countenance upon thee, and give thee peace. Numbers 6: (Holy Bible, KJV) I thank God first of all for this rare opportunity he has given me to continue my professional upliftment in this prestigious school, the School of Veterinary and Biomedical Sciences, James Cook University, Townsville, North Queensland, Australia. I thank Him for the great chance to enjoy bountiful blessings he has bestowed upon me in this country and in advancing my knowledge of my professional career along the course of my study. I thank my scholarship sponsor, the Australian Agency for International Development (AusAID) in providing me financial assistance for without it, I couldn t surely have what I am having now. I also thank and highly acknowledge the exemplary performance of my supervisor, Prof Phillip Summers, for his diligence and ever ready support in the final accomplishment of this work. Not to forget are the helping hands of Chris Coleman and Jeffrey Palpratt during the laboratory works and travels of this research. I thank my mother, family, pastors and friends for prayers and encouragement while I am away from home. Most importantly, I thank God for my wife Angelica, my ever faithful helpmeet. To all of you, I tender this humble piece of work.

6 vi ABSTRACT The aim of this study was to investigate the relationship of maturation of spermatozoa in the epididymis and the ability to bind to oviduct epithelium. It was hypothesized that testicular spermatozoa need to pass through the regions of the epididymis in order to acquire the ability to bind to the oviduct. Spermatozoa were collected from the rete testis and the caput, corpus and cauda epididymides from month-old Large White or Large White x Landrace boars. Boars were first unilaterally castrated and then slaughtered four to five weeks later to obtain the second testicle, epididymis and seminal vesicles. Oviducts were obtained from slaughtered pre-pubertal gilts and explants from the isthmus and ampulla prepared. Spermatozoa were suspended in modified Androhep medium, added to oviduct explants and incubated at 39 0 C in a humidified atmosphere containing 5% CO 2 in air for 15 minutes. The number of spermatozoa attached to 1.25 mm 2 of explant was counted after fixation and staining of explants. The possibility of using oviducts from slaughtered cows rather than porcine oviducts was examined using ejaculated spermatozoa. Significantly more ejaculated spermatozoa bound to the isthmus of gilts than cows hence, porcine oviducts were used in the succeeding experiments. There was a sequential increase in the number of spermatozoa that bound to the oviductal epithelium from the rete testis to the cauda epididymidis (2 ±0.30, 4.36 ±0.53, 9.3 ±1.60 and 15±1.22 for rete testis, caput corpus and caudal spermatozoa on isthmic explants, respectively). Significantly more (P 0.05) spermatozoa, either ejaculated or epididymal, bound to isthmus than ampulla explants (26.33±2.27 and 13.55±1.42 ejaculated spermatozoa on isthmic and ampullary explants, respectively). Incubation in medium containing albumin and asialofetuin which are known to contain mannose and lactose respectively inhibited the binding of epididymal spermatozoa to oviduct explants (3.42±0.56 caudal spermatozoa on isthmic explants in medium with albumin and 14.75±2.02 caudal spermatozoa on isthmic explants in modified Androhep medium).

7 vii The number of spermatozoa from the caput and corpus that bound to oviduct explants significantly (P 0.05) increased after incubation in caudal fluid for 30 minutes (7.52±1.10 and 12.78±1.64 corpus spermatozoa on isthmic explants for modified Androhep and caudal fluid, respectively). This result suggests that caudal fluid has distinct features that directly or indirectly influence the attachment of spermatozoa to oviduct epithelium. The motility of spermatozoa also decreased after incubation in caudal fluid for 30 minutes. This result is not surprising because in vivo, spermatozoa remain in a quiescent state during storage in the cauda epididymidis. Exposure of epididymal spermatozoa to seminal plasma for 30 minutes significantly reduced the number of spermatozoa that bound to oviduct explants while exposure for one minute significantly increased the number of bound caput spermatozoa. Incubation in seminal plasma also caused capacitation of spermatozoa and this is the likely reason for the reduction in the number of bound spermatozoa. There is also the possibility that some components in the seminal plasma may form a coating over the plasma membrane of spermatozoa sufficient to inhibit the expression of pre-existing binding molecules. On the other hand, the seminal plasma may also be a source of binding molecules for immature caput spermatozoa. There was an increase in the motility of spermatozoa after exposure to seminal plasma. In conclusion, this study found that as spermatozoa pass down the epididymis, there is an increase in the number of spermatozoa that bind to oviduct explants. This result was interpreted to mean that the maturation of spermatozoa in the epididymis involves the acquisition of the ability to bind to oviduct epithelium.

8 viii TABLE OF CONTENTS Statement of Access Statement of Sources Declaration of Ethics Statement of the Contribution of Others Acknowledgement Abstract Table of Contents List of Tables List of Figures List of Abbreviations Page No. ii ii iii iv v vi viii xii xiii xvi CHAPTER INTRODUCTION Introduction Working Hypothesis Objectives... 4 CHAPTER REVIEW OF LITERATURE Introduction A General Perspective to the Role of the Epididymis The Fertilising Ability of Epididymal Spermatozoa Comparative Morphology of Spermatozoa from the Caput, Corpus and Cauda Epididymidis Histological Features of the Boar Epididymis Ultrastructural Characteristics of the Boar Epididymis Physiological Correlations The Epididymal Plasma Epididymal secretory proteins Epididymal secretory proteins of the boar and related compounds Secretory enzymes in the epididymal fluid of the boar... 21

9 ix 2.9 Maturation of Boar Spermatozoa in the Epididymis Modifications of the DNA-protein complex Modifications of the plasma membrane Concentration of spermatozoa during the epididymal transit Protection of spermatozoa during storage in the cauda epididymidis The Oviduct Structural anatomy and histology of the oviduct Blood supply to the oviduct The Oviduct Luminal Fluid Binding of Spermatozoa to the Oviduct The role of the sperm reservoir Mechanisms Involved in the Formation of the Sperm Reservoir Physical aspects Carbohydrate recognition Features of the Binding of Spermatozoa to Oviductal Explants Sperm-to-Oviduct Binding in Relation to the Region of the Oviduct, the Stage of the Oestrous Cycle and the Reproductive Status of the Animal Sperm-to-Oviduct Binding between Epididymal and Ejaculated Spermatozoa and between Capacitated and Uncapacitated Spermatozoa The Release of Spermatozoa from the Sperm Reservoir Sperm-Oviduct Binding and the Role of the Seminal Plasma Conclusion CHAPTER MATERIALS AND METHODS Boars Preparation of oviductal explants for the binding assay Preparation of spermatozoa and determination of motility characteristics CASA settings and definitions Co-incubation of spermatozoa and explants Fixation and counting bound spermatozoa Comparison of the binding capacity of ejaculated boar spermatozoa to porcine and bovine oviducts The binding of boar epididymal spermatozoa to porcine oviducts... 52

10 x Comparison of the binding capacity of epididymal boar spermatozoa to oviducts of sows and gilts The binding of epididymal spermatozoa after dilution with either albumin, asialufetuin and modified Androhep solutions The binding of epididymal spermatozoa after incubation in caudal fluid The binding of epididymal spermatozoa to oviductal epithelium after incubation with seminal plasma Determination of the percentage of live and dead spermatozoa Capacitation status of spermatozoa Data presentation and analyses CHAPTER BINDING OF BOAR EPIDIDYMAL SPERMATOZOA TO OVIDUCTAL EPITHELIUM Introduction Binding of ejaculated boar spermatozoa to porcine and bovine oviductal explants Motility of epididymal spermatozoa Binding of boar epididymal spermatozoa to the oviductal epithelium of sows and gilts Developmental influence on the binding ability of boar spermatozoa from the rete testis and the caput, corpus and cauda epididymidis to porcine oviducts Comparison of the binding capacity of epididymal spermatozoa between boars Carbohydrate-binding molecules recognised by epididymal spermatozoa.. 67 CHAPTER EXPOSURE TO CAUDAL EPIDIDYMAL FLUID INCREASES THE BINDING ABILITY OF SPERMATOZOA FROM THE CORPUS AND THE CAPUT EPIDIDYMIDIS Introduction Motility of epididymal spermatozoa after incubation in caudal fluid... 79

11 5.3 Binding of epididymal spermatozoa after incubation in caudal fluid xi CHAPTER INFLUENCE OF SEMINAL PLASMA ON THE BINDING ABILITY OF EPIDIDYMAL SPERMATOZOA Introduction Motility characteristics of epididymal spermatozoa before and after incubation with seminal plasma Capacitation status of spermatozoa before and after incubation in modified Androhep medium or seminal plasma The viability of spermatozoa before and after incubation in modified Androhep medium or seminal plasma The influence of seminal plasma on the binding capacity of epididymal spermatozoa to oviductal epithelium CHAPTER GENERAL DISCUSSION Scope of research project Future research directions Conclusion..107 REFERENCES

12 xii LIST OF TABLES Page No. Table 3.1 Composition of Modified Tyrode s Solution 48 Table 3.2 Composition of the Modified Androhep Solution 51 Table 3.3 Composition of Gill s Haematoxylin stain 52 Table 4.1 Motility characteristics of epididymal spermatozoa immediately after collection 63 Table 5.1 Motility characteristics (mean ± SEM) of epididymal spermatozoa after incubation in either modified Androhep medium or caudal fluid. 80 Table 6.1 Motility characteristics (mean ± SEM) of spermatozoa before and after incubation for 30 minutes in either modified Androhep medium or seminal plasma. 90 Table 6.2 Motility characteristics (mean ± SEM) of spermatozoa before and after incubation for one minute in either modified Androhep medium or seminal plasma. 92

13 xiii LIST OF FIGURES Figure 4.1 Figure 4.2 Page No. Binding of ejaculated porcine spermatozoa to porcine and bovine oviductal and tracheal explants. 62 The mean (+ SEM) percentage of motile spermatozoa from the rete testis and epididymis. 63 Figure 4.3 Binding of epididymal spermatozoa to the isthmus. 64 Figure 4.4 Binding of epididymal spermatozoa to the ampulla. 65 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 Figure 5.1 Binding of boar spermatozoa from the rete testis and epididymis to the isthmus and ampulla. 66 Comparison of the binding of ejaculated and caudal spermatozoa to isthmic and ampullary explants. 66 Comparison between boars in the binding of spermatozoa from the rete testis and epididymis to isthmic explants. 68 Comparison between boars in the binding of spermatozoa from the rete testis and epididymis to ampullary explants. 68 Comparison between the left and the right testicles of boars in the binding capacity of spermatozoa to isthmic explants. 69 Comparison between the left and the right testicles of boars in the binding capacity of spermatozoa to ampullary explants. 69 The binding of epididymal spermatozoa to isthmic explants after incubation of spermatozoa in medium containing either albumin, asialufetuin or modified Androhep medium. 70 The binding of epididymal spermatozoa to ampullary explants after incubation of spermatozoa in medium containing albumin, asialufetuin or modified Androhep medium. 70 The reduction in the motility of spermatozoa from the caput and the corpus after incubation in caudal fluid. 79

14 xiv Figure 5.2 Figure 5.3 Figure 6.1 Figure 6.2 Figure 6.3 Figure 6.4 Figure 6.5 Figure 6.6 Figure 6.7 Figure 6.8 Figure 6.9 Figure 6.10 The influence of incubation of epididymal spermatozoa in caudal fluid on the binding of spermatozoa to isthmic explants. 81 The influence of incubation of epididymal spermatozoa in caudal fluid on the binding of spermatozoa to ampullary explants. 81 The mean (+ SEM) percentage of motile spermatozoa from the rete testis and the epididymis of boars immediately after collection into either modified Androhep medium or seminal plasma. 88 The mean (+ SEM) percentage of motile spermatozoa from the rete testis and the epididymis of boars after incubation for 30 minutes in either modified Androhep medium or seminal plasma. 89 The mean (+ SEM) percentage of motile spermatozoa from the rete testis and the epididymis of boars before and after incubation for one minute in either modified Androhep medium or the seminal plasma. 91 The capacitation status of spermatozoa before incubation in modified Androhep medium (control) or after incubation for 30 minutes in either modified Androhep medium or seminal plasma. 93 The capacitation status of spermatozoa after incubation for one minute in either modified Androhep medium (control) or seminal plasma. 93 The percentage (+SEM) of live spermatozoa before incubation in modified Androhep medium or after incubation for 30 minutes in either modified Androhep medium or seminal plasma. 94 The percentage (+SEM) of live spermatozoa after incubation for one minute in either modified Androhep medium or seminal plasma. 94 Effect of incubation for 30 minutes in either modified Androhep medium or seminal plasma on the binding of spermatozoa from the rete testis and epididymis to isthmic explants. 95 Effect of incubation for 30 minutes in either modified Androhep medium or seminal plasma on the binding of spermatozoa from the rete testis and epididymis to ampullary explants. 96 Effect of incubation for one minute in either modified Androhep medium or seminal plasma on the binding of spermatozoa from the rete testis and epididymis to isthmic explants. 96

15 xv Figure 6.11 Effect of incubation for one minute in either modified Androhep or seminal plasma on the binding of spermatozoa from the rete testis and epididymis to ampullary explants. 97

16 xvi LIST OF ABBREVIATIONS ANOVA ATP BSP camp camp-pka CTC CRISP CTP DNA EAP E-RABP FITC-sZP GPX g HBPs HE HPLC HTM-IVOS kda K + /Na + ratio MAN2B MMPs mrna OECM OGP PAGE PGDS pi pogp SDS TALP analysis of variance adenosine triphosphate bovine seminal plasma protein cyclic Adenosine Monophosphate camp-protein kinase A chlortetracycline cysteine-rich secretory protein cholesterol transferase protein deoxyribonucleic acid oestrous-associated glycoprotein epididymal retinoic acid-binding protein fluorescein-conjugated solubilized zona pellucida glutathione peroxidase g-force/g-load/gravitational acceleration heparin-binding proteins human epididymal protein high performance liquid chromatography Hamilton-Thorne Machine-integrated visual optical system kilodalton potassium/sodium ratio 135 kda alpha-d-mannosidase matrix metalloproteinases messenger ribonucleic acid oviduct epithelial cell monolayer oestrogen-dependent protein polyacrylamide gel electrophoresis prostaglandin D2 synthase isoelectric point porcine oestrogen-dependent protein sodium dodecyl sulfate Tyrode s albumin lactate phosphate